Background/Purpose: The Th17 lineage of CD4 T cells drives autoimmune diseases such as RA, SLE, IBD, and MS. The JAK2/STAT3 signaling pathway is required for Th17 fate and cytokine production. DMARDs such as JAK inhibitors and anti-IL-6 mAbs exploit this pathway to treat autoimmune diseases. While deletion of STAT3 is known to abolish Th17 development, little is known about the role of STAT3 in the maintenance of effector Th17 cells. We generated Th17^∆STAT3 mice that delete STAT3 in Th17 cells after upregulation of IL-17. Thus we examined the functional role of sustained STAT3 expression in Th17 cells during a prototypical Th17-driven autoimmune response in experimental autoimmune encephalomyelitis (EAE).

Methods: IL-17^creROSA26YFP^fl/fl mice were crossed with STAT3^fl/fl mice (Th17^∆STAT3) and compared to age and gender matched IL-17^creROSA26YFP^fl/flSTAT3^fl/+ littermate controls (Th17^ctrl). Mice were immunized with MOG peptide emulsified with CFA and given pertussis toxin on days 0 and 2. Mice were scored daily for disease severity. Cells were isolated from draining lymph nodes (dLN), blood, and/or central nervous system (CNS) and analyzed by flow cytometry and ELISA. For RNA-Seq, YFP⁺ cells were FACS sorted on day 7 post-immunization and RNA-Seq was performed on an Illumina NextSeq500. Results were analyzed using one-way ANOVA or Student’s t-test, except for EAE clinical data analyzed by Mann-Whitney test on each day of scoring.

Results: Sustained STAT3 expression in Th17 cells is critical for Th17 pro-inflammatory functions, as Th17^∆STAT3 mice had reduced incidence and severity of EAE compared to controls. Similarly, the number of CD4 T cells and macrophages were reduced in the CNS. While Th17 cells develop early after immunization in Th17^∆STAT3 mice, there was a decline in Th17 cell numbers after STAT3 deletion, beginning at the peak of LN priming (day 10). We performed RNASeq of day 10 dLN YFP⁺Th17^∆STAT3 and YFP⁺Th17^ctrl cells, which revealed that Th17^∆STAT3 cells had reduced expression of genes associated with cell cycle pathways, and confirmed that YFP⁺Th17^∆STAT3 cells had increased proportions of cells in G0/G1 phase and decreased proportions of cells in S phase and G2/M. Accordingly, we showed that deletion of STAT3 in Th17 cells resulted in an increase in IL-6-mediated phosphorylation of STAT1, which is known to have anti-proliferative effects. Additionally, Th17 cells showed reduced production of the pro-inflammatory cytokines IL-17, GM-CSF, and IFNγ when stimulated with MOG peptide. Surprisingly, cytokine induction by PMA/ionomycin did not require STAT3, suggesting that STAT3 was functioning in a non-transcriptional role. Furthermore, Th17^∆STAT3 cells had reduced mitochondrial membrane potential, which is required for TCR-activated Ca²⁺ flux.

Conclusion: Expression of STAT3 in effector Th17 cells promotes formation and maintenance of pathogenic autoimmune cells by noncanonical functions of STAT3: by reducing STAT1 activation and anti-proliferative effects, and through maintained mitochondrial membrane potential to drive cytokine production in a TCR-dependent manner.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-transcription-factor-stat3-regulates-pathogenic-th17-responses-in-autoimmune-disease-via-noncanonical-roles/